Method of supercooling perishable materials

ABSTRACT

Perishable products, such as food products, can be preserved by cooling to temperatures below their freezing point without ice crystallization. In some embodiments, the perishable product is cooled to temperatures below the freezing point of water while a pulsed electric field and oscillating magnetic field are applied to the product. Apparatus for supercooling perishable products are also provided and include a pulsed electric field generator and an oscillating magnetic field generator.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the National Phase Entry ofPCT/US2014/069402, which claims priority to U.S. Provisional PatentApplication Ser. No. 61/914,270 filed on Dec. 10, 2013, which is hereinincorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under grant numbers2009-65503-05786 and 2014-67017-21650 awarded by the National Instituteof Food and Agriculture. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present application relates generally to the field of preservationof perishable materials, such as food or tissue. Specifically, thepresent application is directed in some aspects to methods and apparatusfor food preservation capable of preserving the freshness of foodproducts while stored at temperatures below the products' freezingpoint. In other aspects the invention can be applied to storing andpreserving other types of perishable materials, such as biologicalproducts, including human organs and other tissue.

Description of the Related Art

The preservation of food products is a critical aspect of public health.Among the various methods of food preservation, chilling foods helps toslow the process of decomposition and the growth of contaminatingmicrobial species. Freezing is one of the most effective methods forensuring the safety of food products and retaining the quality of foodsover long storage periods. In spite of its effectiveness, the process offreezing and thawing poses significant problems with respect to thequality of the foods. For instance, during the freezing process icecrystallization and growth can result in irreversible damage to tissuestructures in meat, fish, fruit and vegetables, such as structuralruptures and changes in osmotic pressure. Other changes observed tooccur in food products during the freezing and thawing process includechanges in the food's sensory properties such as color, taste, andfreshness. Food products subjected to excessively prolonged freezing mayalso experience lipid oxidation, protein denaturation, icerecrystallization, and changes in the moisture content. These degradingeffects on the quality of food products are directly related to thedegree of structural damages to the food products caused by theformation, growth, and distribution of ice crystals within the foodproducts. Such problems associated with freezing food products show theimportance of controlling the formation and growth of ice crystalswithin food products during the storage period.

To overcome these considerable issues, freezing technologies have beendeveloped based on the manipulation of water properties. Most of thesedevelopments are aimed at inducing the quick freezing of water byinstant nucleation or at controlling the size of ice crystals throughthe external application of stress. To this end, several studies haveexamined the supercooling phenomenon under different conditions andtreatments. Supercooled solutions under an electric field have also beeninvestigated. However, none of these studies have assessed how toprolong the supercooled state in foods at freezing temperatures andduring extensive storage periods.

Methods and apparatus for effectively prolonging the supercooled statein foods and other perishable materials, such as organs harvested fortransplantation, would be beneficial and could enable gentler storageand transportation of perishable materials while avoiding many of theproblems associated with freezing and thawing.

SUMMARY OF THE INVENTION

Disclosed herein are methods of achieving and maintaining a supercooledstate in perishable products. Also disclosed herein, are apparatuscapable of supercooling such products according to the methods disclosedherein. These methods and apparatus may be applied to a variety ofperishable materials and/or substances, including food products andbiological tissues, such as organs.

In some aspects, methods of supercooling a perishable product in acontainer are provided. The perishable product may be cooled to atemperature in the range of about 0° C. to about −20° C. while applyingan oscillating magnetic field to the perishable product. In someembodiments a pulsed electric field is also applied while cooling theproduct. Once cooled, the oscillating magnetic field and a pulsedelectric field are applied to the product. The product may be maintainedat the supercooled temperature for a desired period of time. In someembodiments the product may be cooled to about −4° C. to about −7° C. Insome embodiments no ice crystals form in the supercooled product.

The pulsed electric field may be provided as a pulsed squared waveform,and may be provided at a frequency of at least 20 kHz. The oscillatingmagnetic field may have a strength of about 50 to 500 mT, as measured atthe center of the container holding the product.

In some embodiments the perishable product may be selected from foodproducts, organs, tissues, biologics, cell cultures, stem cells,embryos, blood, reactive solutions, and unstable chemical reagents. Insome embodiments the perishable product remains suitable for itsintended purpose after storage at the supercooled temperature.

In some embodiments, methods of preserving a food product at atemperature below the freezing point of the food product are provided.In the methods the food product preferably does not freeze. The foodproduct is cooled to a temperature below its freezing point whileapplying an oscillating magnetic field. In some embodiments a pulsedelectric field is also provided during cooling. The food is maintainedat the temperature below its freezing point while applying a combinationof the oscillating magnetic field and the pulsed electric field.

In some embodiments the food product comprises meat, such as chicken,beef or fish. Is some embodiments the food product comprises vegetables.The food product may be preserved for 24 hours or more, 72 hours ormore, or even more than two weeks.

In some embodiments there is no significant change in one or more of thecolor, drip loss, or tenderness of the food product relative to foodproducts that were not preserved. In some embodiments, one or morecharacteristics of the preserved food product are preserved relative toa food product that was frozen.

The pulsed electric field may be provided as a squared waveform. In someembodiments the squared waveform is provided with a duty cycle of about0.2 to about 0.8. More than one different duty cycle may be providedduring the preservation process. For example, duty cycles of 0.2, 0.5and 0.8 may be provided. The oscillating magnetic field may have astrength of about 50 to 500 mT.

In some embodiments methods of preserving an organ are provided. Theorgan is supercooled to a temperature below 0° C. while applying anoscillating magnetic field. A pulsed electric field may also be providedduring the cooling process in some embodiments. A combination of theoscillating magnetic field and the pulsed electric field are provided tomaintain the organ at the supercooled temperature without freezing. Insome embodiments the organ is maintained at a temperature below 0° C.for more than 24 hours while continuing to apply the pulsed electricfield and oscillating magnetic field. The organ preferably remainsviable for its intended use after storage.

In another aspect, apparatus are provided that can be used forsupercooling perishable products, such as food, organs and tissue. Insome embodiments the apparatus comprises a container for holding one ormore perishable products, a pulsed electric field generator and anoscillating magnetic field generator. The pulsed electric fieldgenerator comprises at least two electrodes arranged to contact theperishable products when they are placed in the container. Theoscillating magnetic field generator is configured to generate anoscillating magnetic field in the container.

In some embodiments the pulsed electric field generator is controlled toprovide a pulsed squared waveform. The pulsed electric field generatormay, for example, be controlled by an insulated-gate bipolar transistor.

In some embodiments the oscillating magnetic field generator comprisesfour solenoid coils at each side of the container.

In some embodiments the apparatus is portable, and may be placed into aseparate freezing apparatus for cooling. In some embodiments theapparatus is part of a refrigerator or freezer. In some embodiments theapparatus comprises elements to reduce the temperature in the containerto a desired temperature, such as in the range of 0° C. to about −20° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the expected temperature profile over time in(A) the normal freezing process and (B) when a supercooled state isattained and prolonged.

FIGS. 2A-C show embodiments of an apparatus for supercooling perishablematerials.

FIGS. 3A and B depict a supercooling compartment portion of an apparatuswith various configurations of contact electrodes according to someembodiments.

FIG. 3A depicts side electrodes (top view) and B) depicts bottomelectrodes (top view).

FIG. 4 displays the stepwise control of OMF and PEF during supercooling.

FIG. 5 displays a schematic view illustrating an embodiment of anapparatus for use in supercooling perishable goods.

FIG. 6A shows the schematic diagram of an apparatus according to someembodiments that was used in the experiments of Example 1.

FIG. 6B provides a three dimensional view of a supercooling cubeapparatus with the PEF and OMF system that can be used, for example, inthe system of FIG. 6A.

FIGS. 7A and 7B show the electrical properties of chicken breasts duringthe freezing process, consisting of (i) supercooling and (ii) phasetransition. FIG. 7A shows the electric current changes during thefreezing process. FIG. 7B shows electrical conductivities of chickenbreasts as a function of temperature. The circled portion indicates thechanges of electric conductivities during the phase transition.

FIGS. 8A and 8B show the modification of the cooling rate of chickenbreasts by strategically combined PEF and OMF treatments. FIG. 8A showsthe square waveform pulsed electric field with duty cycle sequences of0.8 for 300 seconds, 0.5 for 120 seconds, and 0.2 for 90 seconds. FIG.8B shows corresponding temperature profiles of chicken breasts with OMFtreatment only when the duty cycle of 0.2 is applied.

FIG. 9 displays the temperature profiles of chicken breasts stored at−7±0.5° C. The control was fully frozen and reached a temperature of−6.5° C. Samples undergoing the combined PEF and OMF treatments stayedin the supercooling state during the entire testing period.

FIGS. 10A-C show micrographs of chicken breast samples under differentstorage conditions. FIG. 10A shows micrographs for chicken refrigeratedat 4° C. FIG. 10B shows micrographs for chicken frozen at −7° C. andFIG. 10C shows micrographs for chicken supercooled by the PEF and OMFcombination at −7° C.

FIG. 11 shows temperature profiles of supercooled beef at −4° C. for twoweeks. A control beef sample without the application of both of the PEFand OMF was frozen in 2 hours.

DETAILED DESCRIPTION

Improved methods of storage and/or preservation of food products, organsand tissues, and other perishable materials are desired. Disclosedherein are methods of achieving and maintaining a supercooled state insuch perishable products. To control supercooling, a combination ofpulsed electric field (PEF) and oscillating magnetic field (OMF) can beapplied to the perishable materials. It is hypothesized that thecombination of pulsed electric field and oscillating magnetic fieldtechniques significantly influence the mobility of water molecules.Using this combination, stable supercooled materials can be obtainedthrough the continuous reorientation and induced vibration of watermolecules, thereby suppressing the formation of ice.

In some embodiments, methods of supercooling a perishable product, suchas a food product, comprise cooling the perishable product to atemperature below its freezing point while applying a pulsed electricfield and oscillating magnetic field to the perishable product. Thepulsed electric field and oscillating magnetic field are maintainedwhile the product is stored in the supercooled state. In someembodiments the product does not freeze in the supercooled state. Insome embodiments the perishable product is a product that containswater.

In some embodiments the perishable product is first cooled whileapplying an oscillating magnetic field. In some embodiments the pulsedelectric field is not applied at this time. For example, the product canbe supercooled by being placed in an apparatus, as described in moredetail below, while an oscillating magnetic field is applied. Once theproduct has reached a supercool temperature, the pulsed electric fieldis added and the combination of the pulsed electric field andoscillating magnetic field are maintained for as long as the product isto be stored at a supercooled temperature. The time when the oscillatingmagnetic field is on and the pulsed electric field is off can bereferred to as a first phase, while the time when both the oscillatingmagnetic field and pulsed electric field are provided can be referred toas a second phase. One such embodiment is illustrated in FIG. 4. In someembodiments the product is cooled while applying the oscillatingmagnetic field and the pulsed electric field is added when thetemperature has stabilized. In some embodiments the product is cooledwhile applying the oscillating magnetic field and the pulsed electricfield is added when a desired temperature has been reached. For example,the pulsed electric field may be added when a temperature at which theproduct is to be stored has been reached. In some embodiments thetemperature may be between about −1° C. and about −20° C., for exampleabout −7° C. or about −8° C. In some embodiments the product is cooledwhile applying both the oscillating magnetic field and the pulsedelectric field, and both are maintained during storage.

The use of the pulsed electric field and the oscillating magnetic fieldsuppresses the nucleation of ice crystals in the perishable product andthe product attains a supercooled state without freezing. The pulsedelectric field and oscillating magnetic field can be maintained in orderto maintain the perishable product at the supercooled state for anextended period of time, thus maintaining the quality of the product.

In some embodiments, the perishable product is cooled to a selectedtemperature that is less than the freezing temperature of the product.In some embodiments the perishable produce is cooled to a selectedtemperature that is less than the freezing temperature of water, or 0°C. In some embodiments the selected temperature can be between −1° C.and −20° C. Temperatures in the range of −4° C. and −8° C. are commonlyapplied in some embodiments.

In some embodiments the pulsed electric field is applied as a squaredwaveform. In some embodiments, the squared waveform can have a frequencyof about 0 to about 100 kHz. In some embodiments the frequency is about20 kHz or above.

In some embodiments, the squared waveform can be provided with a dutycycle of about 0.1 to about 0.9, more preferably about 0.2 to about 0.8.In some embodiments a duty cycle of 0.2, 0.5 or 0.8 is used. In someembodiments a duty cycle of 0.5 is used.

In some embodiments, more than one duty cycle can be used duringapplication of a pulsed electric field to a product. In some embodimentsa mixed sequence of duty cycles is used. For example, in someembodiments duty cycles of 0.2, 0.5, and 0.8 are used.

When more than one duty cycle is used, each duty cycle is carried outfor a desired length of time. In some embodiments, one or more dutycycles may be applied for a time of from about 1 to about 1000 secondsor more. In some embodiments one or more duty cycles may be applied forabout 50 to 500 seconds. In some embodiments one or more duty cycles areapplied for 90 seconds. In some embodiments one or more duty cycles areapplied for 120 seconds. In some embodiments one or more duty cycles areapplied for about 300 seconds.

In some embodiments one or more duty cycles are applied for the samelength of time. For example, each duty cycle may be carried out for thesame length of time throughout the time that the PEF is provided. Insome embodiments each duty cycle is carried out for a different lengthof time.

A sequence of duty cycles applied for particular lengths of time mayalso be repeated one or more times during application of the PEF. Insome embodiments a PEF with a duty cycle sequence of 0.8, 0.5 and 0.2 isapplied for 300 sec, 120 sec, and 90 sec. respectively, to a perishablematerial. The sequence may be repeated to maintain the perishablematerial in a supercooled state. In some embodiments a duty cyclesequence suitable for supercooling and maintaining a supercooled statein water-containing perishable materials follows the sequence 0.8 for aperiod of 300 seconds, 0.5 for a period of 120 seconds and 0.2 for aperiod of 120 seconds.

In some embodiments, the pulsed electric field has a strength of about0.6 V/cm to about 10 V/cm.

In some embodiments, the oscillating magnetic field has a strength ofabout 50 to 500 milliTesla as measured at the center of the chamberholding the perishable product. In some embodiments the oscillatingmagnetic field has a strength of about 50 to about 150 mT at the centerof the chamber.

The methods described herein may impede ice crystal formation. In someembodiments the perishable product does not freeze during supercoolingor while maintained in a supercooled state. In some embodiments theperishable product is less frozen than the same type of productmaintained at the same temperature for the same amount of time, but thatis not subjected to PEF and OMF as described herein. In someembodiments, no ice crystals are formed within the supercooledperishable product. In some embodiments finer ice crystals are formed inthe perishable product than are formed in the same type of perishableproduct under similar conditions without the application of both of thepulsed electric field and oscillating magnetic field. In someembodiments any ice crystals that may form do not negatively affect thesensory properties and/or intended use of the perishable product.

In some embodiments the perishable product is maintained in asupercooled state for at least 24 hours while continuing to apply thepulsed electric field and oscillating magnetic field. In someembodiments the perishable product is maintained in a supercooled statefor at least 72 hours while continuing to apply the pulsed electricfield and oscillating magnetic field. In some embodiments the perishableproduct is maintained in a supercooled state for at least two weekswhile continuing to apply the pulsed electric field and oscillatingmagnetic field. In some embodiments the perishable product is maintainedin a supercooled state for a month or more. In some embodiments theperishable product does not freeze during the time that it is maintainedin the supercooled state.

As mentioned above, the methods described herein can be applied to thepreservation of food products by maintaining them in a supercooledstate. Thus, in some embodiments, methods of maintaining a food productin a supercooled state comprise supercooling a food product by coolingthe food product to a temperature range below its freezing point, suchas to about 0° C. to about −20° C., or about −4° C. to about −7° C.,while applying an oscillating magnetic field and, in some embodiments apulsed electric field, to the food product as described above. Thetemperature of the food product may be maintained within the range ofabout −4° C. to about −7° C. while applying the combined pulsed electricfield and oscillating magnetic field to the food product. In someembodiments the temperature is maintained for at least 24 hours, or atleast 72 hours. In some embodiments the temperature is maintained fortwo weeks or more.

In some embodiments a food product is preserved such that its qualitiesdo not significantly change from a fresh product. For example, in someembodiments the drip loss from a piece of meat that has been preservedusing the described methods is not significantly different from the driploss in a fresh piece of the meat. Similarly, in some embodiments thereis no significant change in tenderness in the food, such as a piece ofmeat, after the supercooling process. That is, there is not asignificant difference between the tenderness a piece of meat or otherfood that has been treated as described and a fresh piece of the samemeat or food. In some embodiments no structural differences areobservable between a fresh piece of food and a piece of the same foodthat has been preserved as described. In some embodiments there is nochange in color between food (or other product) that has been preservedas described relative to a fresh piece of the same food (or otherproduct).

In addition, the methods described herein can be used to preserve organsor other tissues after harvest and prior to transplantation or otheruse. The organs or tissues may come, for example, from a human oranimal. In some embodiments the organ is a human organ to be used fortransplantation. In this way the quality of the organs or tissues can bemaintained during transport, storage and preparation. In someembodiments, methods of maintaining an organ or other tissue in asupercooled state comprise supercooling the organ or tissue by coolingto a temperature below its freezing point, such as in a range of about0° C. to about −20° C., or about −4° C. to about −7° C., while applyingan oscillating magnetic field (and in some embodiments a pulsed electricfield) to the organ or tissue, essentially as described above. Thetemperature of the organ or tissue may be maintained within the range ofabout −4° C. to about −7° C. for an extended time while applying boththe pulsed electric field and oscillating magnetic field to the organ ortissue. In some embodiments the temperature is maintained for at least24 hours, or at least 72 hours. In some embodiments the temperature ismaintained for two weeks or more. Preferably the organ or tissue remainsviable for its intended use throughout the time that it is maintained ina supercooled state.

In some embodiments the methods may be used to preserve other types ofmaterials such as biologics, cell cultures, stem cells, embryos, blood,reactive solutions, and unstable chemical reagents.

In some embodiments, apparatus that can be used to implement theabove-described methods for supercooling perishable products areprovided. The apparatus typically comprise a container capable ofstoring one or more perishable products; one or more pulsed electricfield generators comprising electrodes positioned to contact the one ormore perishable products when they are placed in the container, and oneor more oscillating magnetic field generators arranged to form anoscillating magnetic field within the interior of the container. Variousembodiments of such apparatus are described in more detail below.

In some embodiments, a refrigerator or other refrigeration or freezingapparatus comprises an apparatus for supercooling food products asdescribed herein (a supercooling apparatus). For example, the apparatusmay be provided as a drawer or compartment within a refrigerator orfreezer.

In some embodiments, an apparatus for supercooling organs comprises acontainer capable of storing one or more organs; a pulsed electric fieldgenerator comprising electrodes arranged to contact the one or moreorgans and inducing an electric field therein; and an oscillatingmagnetic field generator capable of forming an oscillating magneticfield within the interior of the container. The apparatus may be part ofa larger refrigerator or freezer, or other apparatus. In someembodiments the apparatus is portable and may be placed in a largerrefrigerator or freezer for cooling. In some embodiments the apparatusis portable and comprises cooling elements as well as the supercoolingcomponents.

Supercooling

The phenomenon of supercooling may be understood in the context of theice crystallization process. Ice crystallization can be divided intothree subsequent stages; cooling the liquid-state product to itsfreezing point, removing the latent heat of crystallization during thephase transition, and cooling the solid-state product to the finalstorage temperature. In a supercooling process, water cools below thefreezing temperature until a critical nucleation point is reached by theremoval of sensible heat. Ice nucleation is a stochastic process. Thenegative difference between the temperature at this nucleation point andthe standard freezing point is referred to as the degree ofsupercooling. Depending on the physical conditions of the system, i.e.,pressure, temperature, volume, and cooling rate after a certain degreeof supercooling, a sudden nucleation of water crystals occurs.Thereafter, the ice crystals become more compact and undergocrystallization to bulk ice crystals.

FIG. 1 depicts the difference between bulk ice crystallization and aprolonged supercooled state. FIG. 1A shows the temperature profile of acontrol in which an initial cooling brings the control to subzerotemperature which is quickly followed by nucleation, which is indicatedby a sudden increase in temperature. Transformation to the solid crystalform is an isothermal process at the freezing temperature. Oncecrystallization is complete the ice sample cools until its temperaturereaches an equilibrium with its surroundings. In contrast, FIG. 1B showsa sample that is preserved in the supercooled state as described herein.The temperature profile shows that the sample cools to sub-freezingtemperatures, but instead of the nucleation of ice crystals occurring,the sample remains at the sub-freezing temperature in the liquid state.

By preventing water molecules from forming a cluster of a critical sizethat results in ice nucleation, a water-containing material can bemaintained in the supercooled state, thus impeding a phase transition toits frozen state. Alternatively, the formation of ice crystals withinthe material can be controlled, only allowing small ice crystals whichdo not damage the perishable material. Due to the dipole structure ofwater molecules, an electric field can be applied to a water-containingmaterial and the types of waveform, frequency, interpulse duration (dutyratio) and field strength of an electric field can be modified tocontrol the discharge and realignment of water molecules along thedirection of the electric field. Similarly, due to its diamagneticproperties, magnetic fields make an impact on the intermolecularstructure of water.

Cooling of samples in the present methods can be carried out in any of avariety of refrigerators or freezers. In some embodiments a supercoolingapparatus is part of a refrigerator, freezer or other cooling device.For example, the supercooling apparatus may take the form of a drawer orcompartment in a refrigerator, freezer or other cooling device. In someembodiments a supercooling apparatus is placed into a refrigerator,freezer or other cooling device.

Commercial refrigerators and freezers are available and equipped withvariable temperature control enabling the selection of a desiredtemperature. A non-limiting example of a suitable commercial freezer isa General Electric chest freezer FCM7SUWW (GE, Inc., Fairfield, Conn.).

Digital and analog temperature controllers are available to select thetemperature. A suitable non-limiting example of a temperature controlleris the Johnson Controls, Inc., A419 digital temperature controller(Milwaukee, Wis.).

Other methods of cooling that may be used include the use of cryogenicliquids (e.g., N₂) and solid carbon dioxide (dry ice). Other methods ofcooling the perishable materials will be apparent to the skilledartisan.

Pulsed Electric Fields

Pulsed electric fields are created through rapid discharge of electricalenergy within a finite period of time. Such pulses follow a patternknown as a waveform, which represents how an electrical current variesover time. Common waveforms for electrical currents include the squarewave, the sine wave, the ramp, the sawtooth wave, and the triangularwave. In a squared waveform, the amplitude of the wave alternates at asteady frequency between fixed minimum and maximum values, with the sameduration at minimum and maximum. As described elsewhere, in someembodiments a squared waveform is used in applying a PEF to a perishableproduct.

In addition to having a waveform, pulsed electric fields can follow aduty cycle, as discussed briefly above. A duty cycle is the fraction oftime within a given period in which a signal is active. Thus, duty cyclevalues range between 0 and 1. A duty cycle is expressed by therelationship D=t/P, where D is the duty cycle, t is the time the signalis active, and P is the total period over which the signal is delivered.Duty cycles can be programmed to deliver a desired amount of electricalenergy in packets over a given period of time. Such duty cycles canfurther be programmed to follow a sequence in which D and P are variedas the sequence progresses. A non-limiting example of a duty cyclesequence suitable for supercooling and maintaining a supercooled statein water-containing perishable materials follows the sequence 0.8 for aperiod of 300 seconds, 0.5 for a period of 120 seconds, and 0.2 for aperiod of 90 seconds. Another non-limiting example of a duty cyclesequence suitable for supercooling and maintaining a supercooled statein water-containing perishable materials follows the sequence 0.8 for aperiod of 300 seconds, 0.5 for a period of 120 seconds, and 0.2 for aperiod of 120 seconds. The sequence can be repeated for a definedduration of time or indefinitely.

Power supplies used for generating pulsed electric fields are well knownin the art and are commercially available. The power supply can be acapacitor charging power supply with high frequency alternating current(AC). A non-limiting example of a suitable power supply is anintegrated-gate-bipolar-transistor based power supply (IGBT), such asIRAMX20UP60A, available from International Rectifier, El Segundo, Calif.

Electrodes coupled to the power supply are placed such that they aredirectly in contact with the perishable material when it is placed inthe container. Suitable electrode materials include, but are not limitedto stainless steel, titanium, gold, and silver. The electrodes can beformed in a variety of shapes, including but not limited to plates,prongs, and conductive films. The electrodes can further be designedwith multiple holes to enhance the circulation of cold air. Exemplaryelectrodes are illustrated in FIG. 3. Depending, for example, on thetype of food or other perishable material, different types of electrodescan be selected, such as the side electrodes illustrated in FIG. 3A orthe bottom electrodes illustrated in FIG. 3B.

The power supply can provide an input voltage. A suitable, non-limitingpeak-to-peak voltage setting is about 5 V. Suitable, non-limitingelectrical currents provided by the power supply can be up to about 0.04A. The current produced by the power supply can also be characterized bya working frequency. A suitable, non-limiting example of a frequency forpulsed electric fields applied to supercooling is 20 kHz.

Pulsed electric fields can be controlled using function generators.Suitable function generators are commercially available and well-knownin the art. A non-limiting example of a suitable function generator isthe Agilent Technologies 33220A (Santa Clara, Calif.). Functiongenerators control square wave forms with various duty cycles andworking frequencies.

Oscillating Magnetic Fields

An oscillating magnetic field can be applied to a perishable material asdescribed herein. The oscillating magnetic field may be generated, forexample, by using one or more electromagnets or by a combination of anelectromagnet with a permanent magnet. A non-limiting example of asuitable permanent magnet is a NdFeB permanent magnet availablecommercially (N52, DX88-N52, K & J Magnetics, Inc. Jamison, Pa.). Anon-limiting example of a suitable electromagnet functions byalternating the charge and discharge to a magnet wire (e.g., 22 AWG,EIS, Inc., Atlanta, Ga.) coiled to an iron core (e.g., VIMVAR, Ed Fagan,Inc., Franklin Lakes, N.J.). Examples of suitable systems for producingan oscillating magnetic field include, but are not limited to, oneelectromagnet located to one side of the perishable material, twoelectromagnets located on opposite sides of a perishable material, or anelectromagnet and a permanent magnet located on opposite sides of aperishable material. In some embodiments more than one set ofelectromagnets may be utilized. For example, in some embodiments fourelectromagnets are located at each side of the container holding theperishable material.

Like the pulsed electric field, a pulsed magnetic field can be generatedwith a function generator and power supply to the electromagnet.Suitable power supplies are commercially available, and may be, forexample, an IGBT as described above. The oscillating magnetic field isregulated via the function generator through an input voltage, which canrange from 50 to 150 V at a frequency of 1 Hz. A suitable, non-limitingexample of an oscillating magnetic field is a pulse type field with anintensity ranging from −150 mT to 150 mT. Another non-limiting exampleincludes a combined magnetic flux density by permanent magnet andelectromagnet oscillated between 50 to 500 mT per second. A person ofordinary skill in the art will recognize that the magnetic fieldintensity referred to herein is the intensity as measured at the centerof the storage container.

Apparatus for Supercooling Perishable Materials

Apparatus for supercooling perishable materials can be used to preserveperishable materials without significant ice crystallization attemperatures below their respective freezing points.

FIGS. 2A-C show three dimensional view illustrating a non-limitingembodiment of an apparatus 1 for supercooling. The apparatus 1 comprisesa supercooling compartment 2, in which one or more perishable materialscan be placed for supercooling and storage in a prolonged supercooledstate. The size of the cooling compartment can be selected depending onthe type and size of the perishable material to be treated. Anoscillating magnetic field (OMF) generator comprises solenoid coils 6, 7with attached heat sink 3 located on opposing exterior faces 4 and 5 ofthe supercooling compartment 2, and can create an oscillating magneticfield with a defined intensity as measured at the center of the coolingcompartment. The OMF is generated from the solenoid coils 6 and 7respectively located on the opposite faces 4 and 5. In some embodimentsthe apparatus comprises a controller (not shown) for controlling the OMGgenerator. In some embodiments the controls are set to generate an OMFin the container 2 in the range of 50 to 500 mT. The OMF is regulatedthrough input voltage. In some embodiments the input voltage ranges from50 to 150 V at 1 Hz frequency. In some embodiments the OMF strengthmeasured at the center of the cooling compartment 2 ranges from 50-500mT.

The apparatus 1 further comprises a pulsed electric field (PEF)generator which is used to apply the PEF to the perishable material.FIGS. 3A and B shows embodiments of electrodes that can be used tocontact the perishable material. The perishable material is placed incooling compartment 2 and contacted with electrodes 8 and 9. Springs 20and 21 and side electrode supporters 22 and 23 can be used to maintaincontact with the perishable material in the compartment 2. In oneembodiment depicted by FIG. 3A, contact electrodes 8 and 9 arerespectively located on opposing interior faces 11 and 12 of thecontainer 2. In another embodiment depicted in FIG. 3B, patternedcontact electrodes 10, 30 are located on the interior bottom face 13.Vent holes 25 may be provided in one or more faces of the container 2.

The apparatus comprises a controller to control the PEF generator. Insome embodiments the controller is set to deliver an applied PEF as asquared waveform, as described herein. An example of an applied PEFdelivered as a squared waveform at high frequency (20 Hz) with aprogrammed duty cycle is shown in FIG. 4. In this non-limiting example,the cooling decays from an initial food temperature to a supercoolingtemperature during Phase 1 with oscillating magnetic field (OMF) on andPEF off. In Phase 2 (immediately after supercooling is reached), bothOMF and PEF applied with programmed duty cycles are used to maintain thesupercooling temperature of the food product.

FIG. 5 shows an embodiment of a schematic view of apparatus 1. Variabletransformer 14 coupled with an insulated-gate bipolar transistor (IGBT)power supply for OMF 15 provides the power supply to solenoid coils 6and 7 thereby generating an oscillating magnetic field in the center ofcooling compartment 2. Likewise IGBT 16 coupled with variabletransformer 17 provides the PEF to the perishable material via contactelectrodes (not shown). Measurements of temperature and electric fieldstrength may be acquired through a Data Acquisition System (DAQ) 18 viathermocouple 19.

A supercooling apparatus as described herein can be included as part ofa commercial refrigeration or freezing unit. For example, it may be abuilt-in part of a refrigeration or freezing unit. In such aconfiguration, the apparatus can serve as a supercooling storagecompartment. In some embodiments the supercooling storage compartmentmay be removable. In some embodiments, the apparatus can be manufacturedindependently. For example, the apparatus can be portable and can beplaced into a refrigeration or freezing unit. The freezer can be set tothe desired temperature to begin the supercooling process.

EXAMPLES Example 1 Prolonging the Supercooled State in Chicken

1.1 Experimental Procedure

Fresh chicken breast samples were trimmed of all visible connectivetissue and excess fat and cut into 1.5 inch by 1.5 inch by 0.75 inchcubic blocks. All samples were weighed and wrapped in polyethylene (PE)film to avoid superficial dehydration before experiments.

A supercooling system was designed and fabricated consisting of PEF, OMFand real-time temperature, current and voltage measurement apparatus(FIG. 6A). A supercooling cube (1.5 inch by 1.5 inch by 1.5 inch),equipped with one pair of titanium plate electrodes in parallel, wasassembled on the sample holder between a permanent magnet andelectromagnet (FIG. 6B). For efficient cold air circulation, thesupercooling cube was designed with plates having multiple holes.Magnetic forces were applied using a block of NdFeB permanent magnet(N52, Dx88-N52, K&J Magnetics, Inc., Jamison, Pa.; size: 2″ by 2″ by 1″)and an electromagnet by alternating the charge and discharge to a magnetwire (22 AWG, EIS, Inc., Atlanta, Ga.) coiled to iron core (VIMVAR, EdFagan, Inc., Franklin Lakes, N.J.). The supercooling unit fabricatedwith electric and magnetic fields was placed in a commercial chestfreezer (FCM7SUWW, GE, Inc., Fairfield, Conn.).

The oscillating magnetic field was generated by a pulse type of magneticfield with an intensity ranging from −150 mT to 150 mT. The appliedvoltage and pulse duty cycle was 30 V and 0.01, respectively and theapplied frequency was 1 Hz. The combined magnetic flux densities bypermanent magnet and electromagnet were oscillated between 50 to 150 mTper second at the center of the supercooling cube. The pulsed electricfield was generated using an IGBT (IRAMX20UP60A. InternationalRectifier, El Segundo, Calif.) base power supply. Function generators(33220A, Agilent Technologies, Santa Clara, Calif.) were used to controlthe square wave forms with various duty cycles (D) and workingfrequencies. K-type thermocouple wire (PP-K-24S, Omega Engineering,Inc., Stamford, Conn.) aligned at the center of the cell and a dataacquisition unit (DAQ. Agilent 39704A, Agilent Technologies, Inc., PaloAlto, Calif.) was used to monitor and collect he applied voltage andcurrent, and temperature values of samples and air in a freezer. Thedata were scanned and transmitted at intervals of 1 second. The outputsignal was monitored in a digital oscilloscope (Model TDS2014;Tektronix, Beaverton, Oreg.) and the magnetic flux densities between twodifferent magnets was measured using a handheld teslameter (4060.50 AETeslameter, Frederikscn, Inc. Ølgod, Denmark). Freezer temperatures werecontrolled by a digital temperature controller (A419, Johnson Controls,Inc., Milwaukee, Wis.).

A cell was fabricated for measuring electrical conductivities of foodsamples. To measure electrical conductivities of supercooled chickenbreasts, samples were placed and contacted between two electrodes in afreezer, operating at −7±0.5° C. Through the changes in electricalconductivities with temperature, the applied voltage and current weredetermined and tested for the desired cooling temperature profile.Acquired electrical conductivities of the samples were calculated by thefollowing equation:

$\sigma = \frac{LI}{AV}$

Where L is the distance between two electrodes (m), A is the internalcross-sectional area (m²), V is the applied voltage (V) and I is themeasured electric current (A). From the obtained data, collected from 0°C. to the temperature where the phase transition begins, the electricalconductivities of tested food materials were plotted againsttemperature, and the temperature dependence of the measured electricalconductivity was depicted by a linear equation:σ=σ_(ref)+mT

In order to generate a stair-shaped cooling rate, three different dutycycles. 0.2, 0.5 and 0.8 were used for the PEF treatment. The inputvoltage (5 V_(p-p), peak-to-peak voltage) at a frequency of 20 kHz wasset without electroconducting heating even at the maximum duty cycle(0.8); the maximum electric current was estimated to be 0.032 A. Withthe purpose to initiate the supercooling state in samples, the V_(p-p)of 5 V with a duty cycle of 0.5 was applied to the samples until theelectric current reached its minimum value in the supercooling state.The effects of PEF with duty cycle sequences of 0.8, 0.5, and 0.2 on thecooling rate of chicken breasts were explored. The applied periods forthe duty cycle sequence were 300 sec, 120 sec, and 90 sec, respectively.To suppress ice nucleation, the OMF was applied only with PEF duty cycleof 0.2. Using this protocol, the stair-shaped cooling rate controls wererepeated until the temperature of samples reached the freezertemperature, −7±0.5° C. Four experiments were carried out to validatethe reproducibility.

Microstructure Analysis

The microstructures of chicken breast samples under different conditions(refrigeration at 4° C. (control-), freezing at −7° C. (control+), andsupercooling by PEF and OMF combination at −7° C.) were studied using aninverted microscope (Leica-DMIL, Wetzlar, Germany), and changes in cellmorphology were evaluated. The dissected chicken samples were frozen inisopentane at −80° C., and series of 10 μm-thick coronal sections weregenerated with a Leica CM 1900 cryostat (Leica Microsystems BuffaloGrove, Ill.). The structural change by both intra- and extracellular icecrystals was estimated by the cavity size observed in the meatcross-sectional area in the equivalent circular diameter.

Drip Loss

Drip has been used to describe exudates both from frozen thawed meat(drip) and from refrigerated or supercooled meat (weep). The drip losswas measured according to the method previously described by Ngapo etal. (1999. “Freezing and thawing rate effects on drip loss from samplesof pork.” Meat Sci. 53, 149-158), which is incorporated by referenceherein for the limited purpose of disclosing a method for measuring driploss. Fresh meat samples were measured within 30 min after the chickenbreast was cut into chunks; drip loss of PEF and OMF treated and thawedsamples were measured after refrigerated (0-4° C.) storage at 4 hours.Five samples were used for each combination of freezing, thawing andstorage. The samples were cut into six cubes of approximately 0.5 inchlength. Samples were suspended using nylon mesh and centrifuged at 40×gfor 90 min. Drip loss was measured as:

${{Driploss}(\%)} = {\frac{{initialweight} - {finalweight}}{initialweight} \times 100}$Color Measurement

To determine the whether treatments had any negative effects on theappearance of chicken breasts, instrumental color analysis wasconducted. The Hunter L* (lightness), a* (redness-greenness), and b*(yellowness-blueness) values were measured using a color meter (ColorTecPCM, Clinton, N.J.). The net color difference (ΔE) was calculated withthe equation:ΔE=√{square root over ((L ₂ *−L ₁*)²+(a ₂ *−a ₁*)²+(b ₂ *−b ₁*)²)} wherethe subscripts 1 and 2 are referred to as color components before andafter treatment, respectively.Texture Analysis

After cooking in a 75° C. water bath for 30 min, texture was evaluatedby shear force using a TA-XT2 texture analyzer (Stable Micro Systems,Godalming, UK) as described by Barbanti and Pasquini (2005, “Influenceof cooking conditions on cooking loss and tenderness of raw andmarinated chicken breast meat.” LWT—Food Sci. Technol. 38, 895-901,herein incorporated by reference for the limited purpose of textureanalysis method) with a slight modification. The operating parameterswere with a 25 kg maximum cell load applied at a cross-head speed of 5mm/s for 50% cutting distance. Results have been expressed as shearforce (N of sample) and four measurements were performed on each sample.

pH Measurement

The pH value of chicken meat was determined using 5 g samples,homogenized with 5 ml of water. The pH was measured using a digitalpH-meter (Mettler Toledo, Columbus, Ohio) with direct insertion of theprobe electrode after calibration. Measurements of pH were calculatedfrom the average of four replicates.

Lipid Oxidation Measurement

Thiobarbituric acid reactive substances (TBARS) indicate the oxidativechanges in muscle foods during storage. The amounts of TBARS in rawchicken breast samples were determined in triplicates for each testusing the procedure of McDonald and Hultin (1987) and Sayer and others(2001). Samples (1 g) were weighed in plastic bags (ZipLoc, SC Johnson,USA) and homogenized with 10 ml of deionized water. An aliquot of thesample (1 ml) was added to 2 ml of trichloroacetic acid/thiobarbituricacid (TCA/TBA), consisting of 15% TCA (w/v) and 0.375% TBA (w/v) in 0.25M HCl and 3 ml of 2% butylated hydroxytoluene (BHT) (w/v) prepared inethanol and mixed thoroughly. The mixture was vortexed and incubated for15 min in 90° C. of water bath. The sample was cooled at roomtemperature for 10 min and centrifuged for 10 min at 1000×g. Theabsorbance of the resulting supernatant solution was determined at 532nm on a visible spectrophotometer (Thermo Scientific GENESYS20, ThermoFisher Scientific, Inc., Rochester, N.Y.). The TBARS values werecalculated using a molar extinction coefficient of 1.56×10⁵ M⁻¹cm⁻¹ andexpressed as mg malondialdehyde (MDA) per kg of meat sample. For themeasurement of lipid oxidation, four replicates were performed.

Statistical Analysis

The results of this study are presented as the means, standard errorsand analysis of variance (ANOVA) routine to test the significance of thedissimilarities between the means of testing parameters among thetreatments (P<0.05).

1.2 Results

PEF and OMF Combination

Results from the measurement of electric current of raw chicken breastsdemonstrated that its values were changing during the freezing process.The decreasing temperatures of samples triggered a decrease in theamount of flows of electric charge linearly (FIG. 7A). In addition,there was a deflection to the steeper linear trend around −3° C.Significant changes in the electric current values of chicken breastsamples indicate ice nucleation inside of chicken breast samples. Theseobservations were confirmed by the results of electrical conductivitieschanges in the function of temperature. Before ice nucleation occurred,a linear correlation was observed (R²=0.969, FIG. 7(b)). In contrast, nosignificant correlation between electrical conductivities andtemperature was demonstrated after nucleation.

The electrical conductivities of the supercooling state in chickensamples are also given in FIG. 7B. The supercooling was found in thetemperature range −1 to −3° C. before the sudden ice nucleation. Theelectrical conductivities of samples in the supercooling state presentedhigh enough linear correlation (R²=0.969) to be concluded as the samelinear trend as the electrical conductivities of unfrozen state with thelinear function of temperature. The decrease in electricalconductivities in the supercooling temperature range caused a decreasein the electric current of samples and minimum electrical conductivityand electric current in supercooled chicken breasts were estimated to0.580 S/m and 0.024 A, respectively, before nucleation occurred.

For full control of a stable supercooling, the stair-shaped coolingrates were designed using PEF with the sequence of three duty cycles(FIG. 8A). The PEF with duty cycles of 0.8, 0.5 and 0.2 was optimized toapply sequentially in different periods of 300, 120 and 90 seconds,respectively (t₁, t₂, and t₃, respectively, FIG. 8A).

The temperature profiles of the PEF treatment under each of thedifferent duty cycle is shown in FIG. 8B. It can be seen that maximumduty cycle of 0.8 resulted in the constant sample temperature. Thedecrease in duty cycles evoked an increase in cooling rates of chickenbreast samples up to −0.12° C./min. This indicates is that the dutycycle sequence brings about the modification of cooling rates whichleads to a stair-shaped temperature profile.

The combination of PEF and OMF thus successfully prevented icenucleation without any noticeable electrical interference (FIG. 8B).

Effects of Developed PEF and OMF Combination on Extension ofSupercooling

FIG. 9 shows the temperature as a function of time under the combinedPEF and OMF treatments using developed protocol. For comparison of theproposed PEF and OMF treatment protocol, the chicken breasts without anytreatments were employed as a control. The same cooling rates can beseen for controls and the combined PEF and OMF treated samples beforethe controls became frozen. During this period, the effect of thecombined PEF and OMF treatment diverged from the control when theelectric current reached its minimum upon reaching the supercoolingstate (0.024 A). Therefore, it can be concluded that the developed PEFand OMF treatment are based on the interaction of water molecules ratherthan thermal effect.

The PEF and OMF treated samples remained in the supercooled state (noice nucleation), while the controls were fully frozen. Since there is nofreezing point on the chicken samples under PEF and OMF combination, thedegree of supercooling of PEF and OMF treated chicken samples wasestimated by the temperature difference between the freezer temperatureand freezing point for the controls. The mean degree of supercooling ofchicken breasts under PEF and OMF treatment was 5.6±0.2° C., as comparedwith 1.6±1.4° C. for the controls. In all cases, there was no sudden icenucleation to the samples under the developed PEF and OMF treatment.Therefore, the control strategy using developed PEF and OMF combinationwas effective and applicable to maintaining the supercooling state inchicken breast samples.

Effects of Developed PEF and OMF Combination on the Microstructure ofChicken Breasts

The structures of sample tissues in the supercooling state wereillustrated by optical microscopy and micrographs of representativeimages of chicken breasts after different treatments are shown in FIG.10A-C. The micrograph images show that the refrigerated meats maintainedtheir compact fiber tissues and no voids were observed between tissues.On the other hand, when the samples were frozen, the ice crystals wereevident by some voids and distortion of tissues. Even after short termsin the frozen state, the fully frozen samples displayed significantdamages. The equivalent circular diameters of cavities were estimated to204±70 μm. Note that the cavity sizes vary in whole cross-sectional areaand a large number of freeze-cracks were proceeded. The micrographscorresponding to meat in supercooling (FIG. 10C) show no noticeablestructural damage and cell disruption similar to the condition displayedby the refrigerated (unfrozen) sample.

Effects of PEF and OMF Combination on the Qualities of SupercooledChicken Breasts

Quality parameters, including drip loss, color, texture, pH and lipidoxidation, were measured to assess the quality changes on supercooledchicken breast samples. In comparison to the quality factors, thequality values of initial chicken breast samples at 4° C. wereconsidered as controls. Table 1 shows that the quality parameter changesafter different cold temperature storage conditions: fresh, refrigeratedat 4° C. for 12 hours, frozen and supercooled at −7° C. for 12 hours.

TABLE 1 Mean values (±S.D.) of physical and chemical changes overinitial (at 4° C., Control), refrigerated (at 4° C. for 12 hours,Control−), frozen (at −7° C. for 12 hours and thawed at 4° C. for 4hours, respectively, Control+), and supercooled (at −7° C. for 12 hours)chicken breast samples Parameter Initial Refrigerated Frozen SupercooledDrip loss (%)  0.83 ± 0.14^(a)  0.85 ± 0.06^(a)  1.74 ± 0.17^(b)  0.79 ±0.10^(a) Color change N/A  0.35 ± 0.03^(a)  0.32 ± 0.02^(a)  0.33 ±0.04^(a) (ΔE) Texture (N) 27.24 ± 1.68^(a,b) 26.77 ± 1.25^(a) 25.67 ±1.44^(c) 27.30 ± 1.36^(b) pH  6.40 ± 0.01^(a)  6.41 ± 0.01^(a)  6.40 ±0.02^(a)  6.40 ± 0.01^(a) TBARS  0.26 ± 0.03^(a,b)  0.29 ± 0.02^(b) 0.26 ± 0.01^(a)  0.26 ± 0.01^(a) (mg MDA/ kg meat) In each row,dissimilar small letters in each cell indicate a significant differenceat 0.05 levels.

Regarding drip loss, the values were not significantly different forfresh, refrigerated and supercooled chicken breasts. The chicken breastsfrozen at −7° C. showed an increase in drip loss which indicatesmyofibrillar shrinkage and muscle cell damages by the formation of icecrystals. The microstructure of breast meat after frozen storage wasshown in FIG. 10B, and confirms that ice crystal growth during freezingstorage was such that major structural damage occurred in the musclefibers. Therefore, the degree of loss in water holding capacity would beor become similar to the trends that could be observed from the driploss.

The shear force results show that the tenderness of frozen chickenbreasts decreased significantly (P<0.05). The underlying mechanism inthe loss of tenderness is also derived from the breakdown of the musclefibers and the loss of structural integrity caused by ice crystalformation. The formation of extracellular ice crystals disrupts thephysical structure, largely breaking myofibrils apart and resulting inloss of tenderness. In contrast, both the refrigerated and supercooledchicken breasts were as tender as original samples (P>0.05).

The measurements of TBARS in chicken samples demonstrated the amount ofsecondary oxidation product, malondialdehyde (MDA). The TBARS value ofrefrigerated chicken breasts was significantly higher than stored inother conditions. The inhibition of TBARS was estimated at most to be20% by freezing or supercooling as compared to refrigeration and itindicates that subzero temperature storage was effective in reducinglipid oxidation of chicken breasts.

From the foregoing tests, the supercooled chicken breast samplesmaintained the original qualities of a fresh chicken product. Overall,the findings of the current study demonstrate that the strategy of PEFand OMF combination for prolonging the supercooled state is applicableto maintain the original qualities while achieving satisfactory longterm storage of perishable materials.

Example 2 Storage of Meat Products in the Supercooled State

A meat product selected from chicken, beef, pork, fish, or anotheranimal was placed into the cooling compartment of an apparatus asdescribed herein. The apparatus was subsequently transferred to afreezer which had an internal temperature controlled at about −8° C.Upon placement in the freezer, the combined application of PEF and OMFwas initiated. The programmed duty cycles for the PEF and OMF continuedto repeat during the cooling process and throughout the storage period.The meat product was stored in the resulting supercooled state for abouttwo weeks (FIG. 11). The meat product was preserved such that there wasno significant change in color, drip loss or tenderness relative tofresh meat.

Example 3 Storage of a Biological Organ in the Supercooled State

A biological organ is placed into the cooling compartment of anapparatus as described herein. The apparatus is subsequently transferredto a freezer which has an internal temperature controlled at about 0° C.to about −20° C. such as about −7° C. Upon placement in the freezer, theapparatus is used to apply combined PEF and OMF to the organ such thatit is maintained in a supercooled state. The biological organ is storedin the resulting supercooled state and has no significant change instructure or function as a result of the supercooling process.

What is claimed is:
 1. A method of supercooling a perishable product ina container comprising the steps of: cooling the perishable product to atemperature within the range of about 0° C. to about −20° C. whileapplying an oscillating magnetic field to the perishable product; andsimultaneously applying the oscillating magnetic field and a pulsedelectric field to the cooled perishable product while maintaining thetemperature below the freezing point of the perishable product, whereinthe pulsed electric field is applied through a pair of electrodesdirectly in contact with the perishable product.
 2. The method of claim1, wherein the perishable product is a food product.
 3. The method ofclaim 2, wherein the food product is a meat product.
 4. The method ofclaim 1, wherein the perishable product is an organ or body tissue. 5.The method of claim 1, wherein the perishable product is cooled to atemperature within the range of about −4° C. to about −7° C.
 6. Themethod of claim 1, wherein the pulsed electric field is provided as apulsed squared waveform.
 7. The method of claim 6, wherein the squaredwaveform is provided at a frequency of at least 20 kHz.
 8. The method ofclaim 1, wherein the oscillating magnetic field has a strength of about50 to 500 mT as measured at the center of the container.
 9. The methodof claim 1, wherein no ice crystals are formed within the supercooledperishable product.
 10. The method of claim 1, additionally comprisingapplying the pulsed electric field to the perishable product duringcooling.
 11. The method of claim 1, wherein the perishable product isselected from food products, organs, tissues, biologics, cell cultures,stem cells, embryos, blood, reactive solutions, and unstable chemicalreagents.
 12. A method of preserving a food product at a temperaturebelow its freezing point comprising: cooling the food product to atemperature below its freezing point while applying an oscillatingmagnetic field to the food product; and maintaining the food productbelow its freezing point for a desired period of time while applying theoscillating magnetic field and applying a pulsed electric field throughelectrodes contacting the food product.
 13. The method of claim 12,wherein the food product comprises one or more pieces of meat.
 14. Themethod of claim 13, wherein the meat is one or more of chicken, beef andfish.
 15. The method of claim 12, wherein the food product is avegetable.
 16. The method of claim 12, wherein the food product ispreserved for more than 24 hours.
 17. The method of claim 16, whereinthere is no significant change in one or more of the color, drip loss ortenderness of the food product relative to a food product that was notpreserved.
 18. The method of claim 12, additionally comprising applyinga pulsed electric field to the food product during the cooling.
 19. Themethod of claim 12, wherein the pulsed electric field is provided as asquared waveform.
 20. The method of claim 19, wherein the squaredwaveform is provided with a duty cycle of about 0.2 to about 0.8. 21.The method of claim 19, wherein the squared waveform is provided withmore than one duty cycle.
 22. The method of claim 21, wherein the squarewaveform is provided with duty cycles of 0.2, 0.5 and 0.8.
 23. Themethod of claim 12, wherein the oscillating magnetic field has astrength of about 50 to 500 mT.
 24. A method of preserving an organcomprising cooling the organ to below 0° C. while applying anoscillating magnetic field to the organ and subsequently applying apulsed electric field and the oscillating magnetic field to the organ,wherein the pulsed electric field is applied through two contactelectrodes.
 25. The method of claim 24, wherein the pulsed electricfield is provided as a pulsed square waveform.
 26. The method of claim24, wherein the oscillating magnetic field has a strength of about 50 to500 mT.
 27. The method of claim 24, wherein the organ is maintained atthe temperature below 0° C. for more than 24 hours while continuing toapply the pulsed electric field and oscillating magnetic field.
 28. Themethod of claim 1, wherein the pulsed electric field has a strength ofabout 0.6 V/cm to about 10 V/cm.
 29. The method of claim 12, wherein thepulsed electric field has a strength of about 0.6 V/cm to about 10 V/cm.30. The method of claim 24, wherein the pulsed electric field has astrength of about 0.6 V/cm to about 10 V/cm.